The present invention relates to a novel tetrodotoxin resistant sodium channel and related nucleotides, as well as screening assays for identifying agents useful in treating acute or chronic pain or other hyperexcitability states.
A. Sodium Channels
Voltage-gated sodium channels are a class of specialized protein molecules that act as molecular batteries permitting excitable cells (neurons and muscle fibers) to produce and propagate electrical impulses. Voltage-gated Na+ channels from rat brain are composed of three subunits, the pore-forming xcex1 subunit (260 KDa) and two auxiliary subunits, xcex21 (36 KDa) and xcex22 (33 KDa) that may modulate the properties of the xcex1-subunit; the xcex1 subunit is sufficient to form a functional channel that generates a Na current flow across the membrane [references 1,2 as cited below]. Nine distinct xcex1 subunits have been identified in vertebrates and are encoded by members of an expanding gene family [3 and references therein, 4-6] and respective orthologues of a number of them have been cloned from various mammalian species including humans. Specific xcex1 subunits are expressed in a tissue- and developmentally-specific manner [7,8]. Aberrant expression patterns or mutations of voltage-gated sodium channel xcex1-subunits underlie a number of human and animal disorders [9-13].
Voltage-gated sodium channel xcex1-subunits consist of four domains (D1-4) of varying internal homology but of similar predicted structure, connected by three intracellular loops (L1-3). The four domains fold to form a channel that opens to both the cytoplasm and the extracellular space via a pore. The pore opens and closes depending upon the physiological state of the cell membrane.
Each domain consists of six transmembrane segments (S1-6) that allow the protein to weave through the membrane with intra- and extracellular linkers. The linkers of S5-S6 segments of the four domains contain sequences that line the pore of the channel, and a highly conserved subset of amino acids that acts as a filter to selectively allow sodium ions to traverse the channel pore into the cytoplasm, thus generating an electric current. The amphiphatic S4 segment, in each of the four domains, rich in basic residues repeated every third amino acid, acts as a voltage sensor and undergoes a conformational change as a result of the change in the voltage difference across the cell membrane. This in turn triggers the conformational change of the protein to open its pore to the extracellular Na+ ion gradient.
In most of the known voltage-gated sodium channel xcex1-subunits the channels close and change into an inoperable state quickly (inactivate) within a few milliseconds after opening of the pore (activation); SNS-type channels, on the other hand, inactivate slowly and require a greater voltage change to activate. L3, the loop that links domains D3 and D4, contains a tripeptide which acts as an intracellular plug that closes the pore after activation, thus inducing the channel to enter the inactive state. After inactivation, these channels further undergo conformational change to restore their resting state and become available for activation. This period is referred to as recovery from inactivation (repriming). Different channels reprime at different rates, and repriming in SNS is relatively rapid.
Based on amino acid similarities, the voltage-gated sodium channel family has been further subdivided into two subfamilies [14]. Eight of the nine cloned channels belong to subfamily 1. They share many structural features, particularly in their S4 transmembrane segments. However, some of them have been shown to have distinct kinetic properties of inactivation and repriming. Only a single channel of subfamily 2, also referred to as atypical channels, has been identified in human, rat and mouse tissues. This subfamily is primarily characterized by reduced numbers of basic residues in its S4 segments, and thus is predicted to have different voltage-dependence compared to subfamily 1. The physiological function of subfamily 2 channels is currently unknown because its electrophysiological properties have not yet been elucidated.
The blocking of voltage-gated sodium channels by tetrodotoxin, a neurotoxin, has served to functionally classify these channels into sensitive (TTX-S) and resistant (TTX-R) phenotypes. Two mammalian TTX-R channels have so far been identified, one specific to the cardiac muscle and to very limited areas of the central nervous system (CNS) and the second, SNS, is restricted to peripheral neurons (PNS) of the dorsal root ganglia (DRG) and trigeminal ganglia. Specific amino acid residues that confer resistance or sensitivity to TTX have been localized to the ion selectivity filter of the channel pore. The SNS channel is also described in International Patent Application WO 97/01577.
B. Role of Sodium Channels in Disease States
Because different Na+ channel xcex1-subunit isotypes exhibit different kinetics and voltage-dependence, the firing properties of excitable cells depend on the precise mixture of channel types that they express. Mutants of the cardiac and skeletal muscle xcex1-subunit have been shown to cause a number of muscle disorders. Some examples are as follows: A change of a single basic amino acid residue in the S4 of the skeletal muscle channel is sufficient to change the kinetic properties of this channel and induce a disease state in many patients. A tripeptide deletion in L3 of the cardiac channel, proximal to the inactivation gate, induces a cardiac disorder called Long QT syndrome. A single amino acid change in the S5-S6 linker of domain 1 of Scn8a, the region lining the pore of the channel, causes the mouse mutant xe2x80x9cjoltingxe2x80x9d. The total loss of this channel by a different mutation causes motor end plate xe2x80x9cmedxe2x80x9d disease in mice. This mutation is characterized by loss of motor neuron stimulation of the innervated muscle.
C. Sodium Channels and Pain
Axonal injury (injury to nerve fibers, also called axons) can produce chronic pain (termed neuropathic pain). A number of studies have demonstrated altered excitability of the neuronal cell body and dendrites after axonal injury [15-17], and there is evidence for a change in Na+ channel density over the neuronal cell body and dendrites following axonal injury [18-20]. The expression of abnormal mixtures of different types of sodium channels in a neuronal cell can also lead to abnormal firing [13], and can contribute to hyperexcitability, paresthesia or pain.
Recent studies from our group on rat sensory DRG neurons have demonstrated a dramatic change in the expression profile of TTX-R and TTX-S currents and in a number of mRNA transcripts that could encode the channels responsible for these currents in DRG neurons following various insults [21-23]. We have, for example, shown an attenuation of the slowly inactivating, TTX-R current and simultaneous enhancement of the rapidly inactivating, TTX-S Na+ currents in identified sensory cutaneous afferent neurons following axotomy [21]. We also have shown a loss of TTX-S, slowly repriming current and TTX-R current and a gain in TTX-S, rapidly repriming current in nociceptive (pain) neurons following axotomy [22], down-regulation of SNS transcripts and a simultaneous up-regulation of xcex1-III Transcripts [23]. Also associated with axotomy is a moderate elevation in the levels of xcex1I and xcex1II mRNAs [24]. These changes in the sodium channel profile appear to contribute to abnormal firing that underlies neuropathic pain that patients suffer following axonal injury.
Inflammation, which is also associated with pain (termed inflammatory pain), also causes alteration in the sodium current profile in nociceptive DRG neurons. Inflammatory modulators up-regulate TTX-R current in small C-type nociceptive DRG neurons in culture [25,26]. The rapid action of these modulators suggests that their action include posttranslational modification of existing TTX-R channels. We have now determined that inflammation also increases a TTX-R Na+ current and up-regulates SNS transcripts in C-type DRG neurons [58]. This data suggests that changes in the sodium current profile contribute to inflammation evoked-pain.
D. Therapies for Chronic Pain
A variety of classes of drugs (anticonvulsants such as phenytoin and carbamazepine; anti-arrhythmics such as mexitine; local anesthetics such as lidocaine) act on Na+ channels. Since the various Na+ channels produce sodium currents with different properties, selective blockade or activation (or other modulation) of specific channel subtypes is expected to be of significant therapeutic value. Moreover, the selective expression of certain xcex1-subunit isoforms (PN1, SNS, NaN) in specific types of neurons provides a means for selectively altering their behavior.
Nociceptive neurons of the DRG are the major source of the PNS TTX-R Na+ current. Thus, the Na+ channels producing TTX-R currents provide a relatively specific target for the manipulation of pain-producing neurons. The molecular structure of one TTX-R channel in these DRG neurons, SNS, has been identified but, prior to our research, it has not been determined whether there are other TTX-R channels in these neurons. If such channels could be identified, they would be ideal candidates as target molecules that are preferentially expressed in nociceptive neurons, and whose modulation would attenuate pain transmission.
The present invention includes an isolated nucleic acid which encodes a voltage gated Na+ channel that is preferentially expressed in dorsal root ganglia or trigeminal ganglia (the NaN channel). (In our preceding U.S. Provisional Application No. 60/072,990, this NaN channel was referred to by its previous name xe2x80x9cNaX.xe2x80x9d) In a preferred embodiment, the isolated nucleic acid comprises the sequence shown in FIG. 1, FIG. 7A, FIG. 8A, FIG. 11A (SEQ ID NOS. 1, 4, 6 and 41 respectively), allelic variants of said sequences or nucleic acids that hybridize to the foregoing sequences under stringent conditions.
In another embodiment, the invention includes an expression vector comprising an isolated nucleic acid which encodes the voltage gated Na+ channel that is preferentially expressed in dorsal root ganglia or trigeminal ganglia either alone or with appropriate regulatory and expression control elements. In a preferred embodiment, the expression vector comprises an isolated nucleic acid having the sequence shown in FIG. 1, FIG. 7A, FIG. 8A, FIG. 11A, allelic variants of said sequences or nucleic acids that hybridize to the foregoing sequences under stringent conditions.
The present invention further includes a host cell transformed with an expression vector comprising an isolated nucleic acid which encodes a voltage gated Na+ channel that is preferentially expressed in dorsal root ganglia or trigeminal ganglia with appropriate regulatory and expression control elements. In a preferred embodiment, the expression vector comprises an isolated nucleic acid having the sequence shown in FIG. 1, FIG. 7A, FIG. 8A, FIG. 11A, allelic variants of said sequences or nucleic acids that hybridize to the foregoing sequences under stringent conditions.
The present invention also includes an isolated voltage gated Na+ channel that is preferentially expressed in dorsal root ganglia or trigeminal ganglia. In a preferred embodiment, the channel has the amino acid sequence of FIGS. 2, 7B, 8B or 11B (SEQ ID NOS. 3, 5, 8 and 42, respectively), or is encoded by a nucleic acid having the sequence shown in FIGS. 1, 7A, 8A or 11A, allelic variants of said sequences or nucleic acids that hybridize to the foregoing sequences under stringent conditions. Peptide fragments of the channel are also included.
Another aspect of the invention is a method to identify an agent that modulates the activity of the NaN channel, comprising the steps of bringing the agent into contact with a cell that expresses the Na+ channel on its surface and measuring depolarization, or any resultant changes in the sodium current. The measuring step may be accomplished with voltage clamp measurements, by measuring depolarization, the level of intracellular sodium or by measuring sodium influx.
Another aspect of the invention is a method to identify an agent that modulates the transcription or translation of mRNA encoding the NaN channel. The method comprises the steps of bringing the agent into contact with a cell that expresses the Na+ channel on its surface and measuring the resultant level of expression of the Na+ channel.
The invention also includes a method to treat pain, paraesthesia and hyperexcitability phenomena in an animal or human subject by administering an effective amount of an agent capable of modulating, such as by inhibiting or enhancing, Na+ current flow through NaN channels in DRG or trigeminal neurons. The method may include administering an effective amount of an agent capable of modulating the transcription or translation of mRNA encoding the NaN channel.
Another aspect of the invention is an isolated nucleic acid that is antisense to the nucleic acids described above. In a preferred embodiment, the antisense nucleic acids are of sufficient length to modulate the expression of NaN channel mRNA in a cell containing the mRNA.
Another aspect of the invention is a scintigraphic method to image the loci of pain generation or provide a measure the level of pain associated with DRG or trigeminal neuron mediated hyperexcitability in an animal or human subject by administering labeled monoclonal antibodies or other labeled ligands specific for the NaN Na+ channel.
Another aspect of the invention is a method to identify tissues, cells and cell types that express the NaN sodium channel. This method comprises the step of detecting NaN on the cell surface, or en route to the cell surface, or the presence of NaN encoding mRNA.
The present invention further includes a method of producing a transformed cell that expresses an exogenous NaN encoding nucleic acid, comprising the step of transforming the cell with an expression vector comprising an isolated nucleic acid having the sequence shown in FIGS. 1, 7A, 8A or 11A, allelic variants of said sequences or nucleic acids that hybridize to the foregoing sequences under stringent conditions, together with appropriate regulatory and expression control elements. The invention also includes a method of producing recombinant NaN protein, comprising the step of culturing the transformed host under conditions in which the NaN sodium channel or protein is expressed, and recovering the NaN protein.
The invention also includes an isolated antibody specific for the NaN channel or polypeptide fragment thereof. The isolated antibody may be labeled.
Another aspect of the invention includes a therapeutic composition comprising an effective amount of an agent capable of decreasing rapidly repriming sodium current flow in axotomized, inflamed or otherwise injured DRG neurons or in normal DRG neurons that are being driven to fire at high frequency. The invention also includes a method to treat acute pain or acute or chronic neuropathic or inflammatory pain and hyperexcitability phenomena in an animal or a human patient by administering the therapeutic composition.
The present invention also includes a method to screen candidate compounds for use in treating pain and hyperexcitability phenomena by testing their ability to alter the expression or activity of an NaN channel mRNA or protein in axotomized, inflamed or otherwise injured DRG neurons.